Technical features

When untreated lymphoid cells from two different individuals are cultured together, the resulting MLR is bidirectional. Thus, with a mixture of homozygous MHC-incompatible a and b lymphoid cells, the response involves a T cells reacting to b alloantigens plus b T cells responding to a antigens. In accordance with the classic laws of transplantation immunity, only a T cells proliferate when homozygous a lymphoid cells are cultured with heterozygous {axb)F, cells. Bidirectional responses occur with a mixture of (axb)Fi and (axe)F, cells.

To ensure that the MLR is unidirectional, one of the two populations of lymphoid cells is generally treated with mitomycin C (an inhibitor of DNA synthesis) or subjected to irradiation. Such treatment of the stimulator population destroys or inactivates T cells in this population but does not interfere with the immunogenicity of the treated cells. As discussed below, the stimulatory function of lymphoid cells is restricted to specialized antigen-presenting cells (APCs). These cells are highly radioresistant and do not divide in culture.

As with any assay system conducted in vitro, the magnitude of the MLR depends critically upon the quality of the tissue culture conditions used. To obtain high responses it is essential to use fully viable cells and a tissue culture medium containing all essential nutrients; addition of serum (1-10%) and 2-mercaptoethanol is usually found to be critical. As responder cells, any population containing a high proportion of T cells can be used. Buffy coat white-blood cells are employed as responders (and stimulators) for MLR in humans. For rodents, lymph node (LN) cells give near-optimal data, although most investigators tend to use spleen cells. MLRs with spleen cells, although adequate, are lower than with LN cells (because of the lower content of T cells in spleen) and are often associated with high background responses, i.e. responses with syngeneic stimulators. The best results are obtained with purified T cells as responder cells.

Typical MLRs are usually conducted in 96-well plates in 200 pi volumes, using 5 x 104 to 2x10' responder cells and 5x10^ allogeneic versus syngeneic stimulator cells. Proliferative responses are quantitated by adding |'H ¡thymidine f'HTdR) (1 pCi per well) to the cultures after 1-6 days and harvesting the cultured cells 6-24 hours later to measure 3HTdR incorporation. With low doses of responder cells, e.g. 5xl04, responses generally reach peak levels on day 6 or later. With higher doses of responders, peak responses occur earlier.

For rodents, the magnitude of the MLR depends critically upon the health of the animal colony. Rodents with chronic viral infections generally give rather low MLR, probably because of toxic cytokines or other mediators released by infected macrophages. In this situation, addition of the prostaglandin inhibitor, indomethacin, to the culture often improves the results.

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